targeting blockage of stat3 in hepatocellular carcinoma ...ifn derived from stat3-blocked...

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Targeting Blockage of STAT3 in Hepatocellular CarcinomaCells Augments NK Cell Functions via ReverseHepatocellular Carcinoma–Induced Immune Suppression

Xiaoxia Sun1, Qiangjun Sui1, Cai Zhang1, Zhigang Tian1,2, and Jian Zhang1

AbstractSTAT3 is an important transcriptional factor for cell growth, differentiation, and apoptosis. Although evi-

dence suggests a positive role for STAT3 in cancer, the inhibitory effects of tumor STAT3 on natural killer (NK)

cell functions in human hepatocellular carcinoma are unclear. In this study, we found that blocking STAT3 in

hepatocellular carcinoma cells enhanced NK-cell antitumor function. In the case of STAT3-blocked hepatocel-

lular carcinoma cells, NKG2D ligandswere upregulated, which promoted recognition byNK cells. Importantly,

the cytokine profile of hepatocellular carcinoma cells was altered; in particular, TGF-b and interleukin 10 (IL-10)expressionwas reduced, and type I interferon (IFN)was induced, thus facilitatingNK-cell activation. Indeed, the

cytotoxicity of NK cells treated with supernatant from STAT3-blocked hepatocellular carcinoma cells was

augmented, with a concomitant elevation of molecules associated with NK cytolysis. Further experiments

confirmed that the recovery of NK cells depended on the downregulation of TGF-b and upregulation of type IIFN derived from STAT3-blocked hepatocellular carcinoma cells. These findings demonstrated a pivotal role

for STAT3 in hepatocellular carcinoma-mediated NK-cell dysfunction, and highlighted the importance of

STAT3 blockade for hepatocellular carcinoma immunotherapy, which could restore NK-cell cytotoxicity in

addition to its direct influence on tumor cells. Mol Cancer Ther; 12(12); 2885–96. �2013 AACR.

IntroductionThe tumor microenvironment is a highly complex

milieu, consisting of cancer cells, stromal tissue (immunecells, fibroblasts, myofibroblasts, cytokines, and vasculartissue), as well as the surrounding extracellular matrix (1).These components not only promote tumor progressionand metastasis, but also induce immunosuppression andprotect tumor cells fromhost immuneattack (2).Asamajorbarrier to cancer progression, the immune system controlstumor elimination; however, it is modulated by factors inthe tumor microenvironment. Accumulating evidenceshows that natural killer (NK) cells play a critical role intumor immunosurveillance and act as the first line of thedefense against tumor cells. Enhanced NK-cell infiltrationof the tumor site will recruit host immune defense factorsand inhibit tumorigenesis. Nevertheless, in many patients

with tumors, such as breast, lung, colorectal, and livercancers, both peripheral blood NK cells and tumor-asso-ciatedNKcells exhibit an alteredphenotype andprofounddefects in their degranulation ability and interferon gam-ma (IFN-g) production (3–5). Inaddition, it is indicated thatimmunomodulatory factors in the tumor microenviron-ment, including interleukin 4 (IL-4), IL-10, TGF-b, andindoleamine 2,3-dioxygenase (IDO), contribute to thedecreased expression of stimulatory receptors (NKG2D,DNAM-1, NKp30, and NKp44) and impaired secretion ofcytotoxic proteins (perforin, granzymes) and IFN-g byNKcells, which limit the NK-cell–mediated antitumor effectandpromote tumor immune evasion (1, 6, 7). Therefore, anideal antitumor immunotherapy should improve thetumor microenvironment and augment NK-cell functionswhen applied in the clinic.

STAT3 is a key transcriptional regulator of genes thatcontrol cell growth and differentiation, including bcl-xl,cyclinD1, c-myc,mcl-1,VEGF, IL-10, TGF-b and survivin (8–10). Constitutive activation of STAT3 has been detected ina number of human primary tumors and cancer cell lines,indicating that inhibition of activated STAT3 would sup-press proliferation and induce apoptosis of cancer cells(11–13). In addition, aberrant activation of STAT3 con-tributes to tumor immune evasion via restraining antitu-mor immunity (14–16). Inactivation of STAT3 signaling inhematopoietic cell-specific conditional knockout (CKO)mice (17),or by the use of pharmacologic inhibitors, suchas JSI-124 (18) or CPA-7 (17), resulted in enhanced

Authors' Affiliation: 1Institute of Immunopharmacology & Immunothera-py, School of Pharmaceutical Sciences, Shandong University, Shandong;and 2School of Life Sciences, University of Science and Technology ofChina, Anhui, China

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Jian Zhang, Institute of Immunopharmacology &Immunotherapy, School of Pharmaceutical Sciences, Shandong Univer-sity, 44 Wenhua West Road, Jinan 250012, China. Phone: 86-531-8838-3781; Fax: 86-531-8838-3782; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-1087

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on April 2, 2021. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst October 9, 2013; DOI: 10.1158/1535-7163.MCT-12-1087

http://mct.aacrjournals.org/

antitumor immune responses through the activation ofvarious immune cells such as dendritic cells (DCs) andcytotoxic T lymphocyte (CTL), and inactivation of regu-latory T cell (Tregs; refs. 17, 18).

It has been reported that the inactivation of STAT3 intumor cells favored NK-cell- and T-cell–mediated anti-tumor immune responses (19–21); however, whether NKcell function can be augmented by blocking STAT3 inhuman hepatocellular carcinoma cells has not been fullyinvestigated. In this study, we focus on NK-cell–medi-ated antitumor immune responses to STAT3-blockedhepatocellular carcinoma cells. The results showed thatblocking STAT3 enhanced NK-cell antitumor functionnot only by promoting recognition by NK cells, but alsoby elevating the expression of molecules associated withNK-cell cytolysis. Furthermore, both TGF-b reductionand type I IFN production by STAT3-blocked hepato-cellular carcinoma cells were responsible for NK-cellactivation. Collectively, our studies demonstrated a piv-otal role for STAT3 aberrant signaling in hepatocellularcarcinoma-mediated NK cell dysfunction; in addition,blocking STAT3 in hepatocellular carcinoma cells notonly promoted hepatocellular carcinoma apoptosis, butalso improved NK cell cytotoxicity, which limit/preventtumor cell escape from antitumor immunity.

Materials and MethodsCell lines and primary cell cultures

Hepatocellular carcinoma cell lines HepG2 (The CellBank of Type Culture Collection of the Chinese Academyof Sciences, TCHu 72), H7402 (Institute of Basic MedicalSciences, Shandong Academy of Medical Science, China),and PLC/PRF/5 (kindly supplied by Dr. Qu Xianjun,Department of Pharmacology, Shandong University)were grown in RPMI-1640 medium (GIBCO/BRL) sup-plemented with 10% FBS. The human NK cell line NKLwas generously provided by Dr. Jin Boquan (FourthMilitary Medical University, China) and cultured inRPMI-1640, containing 10% FBS and 100 U/mL rhIL-2(Changsheng, Changchun, China). The human NK cellline NK-92 was purchased from the American Type Cul-ture Collection (CRL-2407) and maintained in a-Mini-mum Essential Medium (MEM; GIBCO/BRL) supple-mented with 12.5% horse serum (GIBCO), 12.5% FBS,100 U/mL rhIL-2, 0.1 mmol/L b-mercaptoethanol and0.02 mmol/L folic acid. All cells were cultured in ahumidified incubator with 5% CO2 at 37

�C according tothe vendor’s instruction, and used within 6 months afterreceipt or resuscitation. Healthy peripheral blood mono-nuclear cells (PBMC) were isolated by Ficoll densitygradient centrifugation and cultured in RPMI-1640 con-taining 10% FBS and 100 U/mL rhIL-2. Informed consentwas provided by all participants enrolled in this study.

STAT3 decoy/scramble oligodeoxynucleotideUsing phosphorothioate chemistry, sense and antisense

strands of STAT3decoy or scramble oligodeoxynucleotide

(ODN) were synthesized by the Expedite Nucleic AcidSynthesis System (Takara Biotechnology). STAT3 decoyODN sequence was 50-CATTTCCCGTAAATC-30, 30-GTAAAGGGCATTTAG-50 and scramble ODN sequencewas 50-CATCTTGCCAATATC-30, 30-GTAGAACGGT-TATAG-50. The sense and antisense strands wereannealed and purified by high-performance liquid chro-matography (HPLC; ref. 22).

Cell transfectionTransient transfections were carried out with Lipofec-

tamine 2000 (Invitrogen) according to the manufacturer’sinstructions. One day before transfection, human livercancer cells were seeded to ensure greater than 90%confluence. After being washed with PBS, cells weretransfected with Lipofectamine 2000/decoy ODN orLipofectamine 2000/scramble ODN. For transfection ofoligonucleotides, 25 nmol/L STAT3 decoy or scrambleODN were used, with an ODN (mg) to Lipofectamine2000 (mL) ratio of 1: 2.5.

NK cells treated with the supernatant from tumorcells

After transfection with ODN for 6 hours, the transfec-tionmediumwas removed, and hepatocellular carcinomacells were washed with 1� PBS to remove residual ODN.Hepatocellular carcinoma cells were then plated at adensity of 1 � 104 cells/well in 96-well plates, and cul-tured in fresh medium (containing 10% FBS) for a further24 hours. Supernatantswere collected and cells anddebrisremoved by centrifugation. NK cells were cultured in thesoluble supernatant for 12 hours with ratio of supernatantto medium (v/v) of 1:1.

Flow cytometryThe phenotype of cells was analyzed by flow cytome-

try. The fluorescence-conjugated antibodies used in thisstudy are described in Supplementary Table S1. Cellswere incubated with fluorescence-conjugated antibodiesfor 30 minutes at 4�C. For detection of intracellular cyto-kines, cells were stimulated for 4 hours with monensin(6 mmol/L) and ionomycin (1 mg/mL; Sigma ChemicalCo.) in a 37�C, 5%CO2 incubator, and thenwashed, fixed,and permeabilized. Cells were then stained with a satu-rating amount of the fluorescence-conjugated antibodiesfor 1 hour at 4�C. After washing with PBS, stained cellswere acquired using a FACSCalibur system (BD Bios-ciences) and analyzed with WinMDI 2.0 software.

RNA isolation and quantitative real-time PCRQuantitative real-time PCR (qRT-PCR) was performed

according to the manufacturer’s instructions. Briefly, totalRNA was isolated using the TRIzol Reagent (Invitrogen).Thequality andconcentrationof theRNAweredeterminedbyspectrophotometricmeasurementof theA260/A280 ratio.cDNA was synthesized using the M-MLV reverse tran-scriptase (Invitrogen). qRT-PCR was performed usingthe SYBR Green Real-time PCR Kit (TOYOBO) on an iQ5

Sun et al.

Mol Cancer Ther; 12(12) December 2013 Molecular Cancer Therapeutics2886




Real-Time PCR detection system (Bio-Rad). RelativemRNA expression levels of the gene of interest were cal-

culatedusing the2�DDCt method. Theprimers aredescribedin Supplementary Table S2.

Luciferase reporter gene assayFor the reporter gene assay, hepatocellular carcinoma

cells were plated at a density of 1� 104 cells per well in 96-well plates (Costar), and transiently cotransfected withpGL3-TGFb1-Promoter-Luciferase or pGL3-IL10-Promot-er-Luciferase (200 ng/well) and STAT3-decoy or scrambleODN in the presence of Lipofectamine 2000, whereas theRenilla expression vector pRLTK (20 ng/well, Promega)was cotransfected to normalize the transfection efficiency.After 24 hours, cells were washed, lysed, and a dual-GloLuciferase assay system (Promega) was used to determineluciferase activity according to themanufacturer’s instruc-tions. The ratio of firefly and Renilla luciferase activityassociated with pGL3-TK-Luciferase transfection was setas 1.

ELISATwenty-four hours after transfection with decoy ODN

or scramble ODN, the supernatant from human livercancer cell culture was collected and centrifuged toremove cells and debris. The concentrations of TGF-b,IL-8, and IL-10 were determined using ELISA kits (ExCellBiology, Inc). To determine the level of IFN-g produced byNK cells, NK-92 cells were incubated both in the presenceand absence of the supernatant from hepatocellular car-cinoma cells. After 12 hours, these cells were centrifuged,resuspended in fresh medium, and cultured for 48 hoursbefore the concentration of IFN-g was measured using anELISA kit.

Cytokines and blocking antibodiesAnti-hULBP3 monoclonal antibody (mAb; 30 mg/mL;

R&D systems, Inc.), recombinant human TGF-b1 (2.5ng/mL, PeproTech), anti-human LAP (TGF-b1) anti-body (2 mg/mL, R&D systems, Inc.), IFN-a and IFN-b(400 U/mL, Changsheng Life Sciences Ltd), and anti-IFNAR mAb (10 mg/mL, PBL) were used in neutraliza-tion assays.

Cell viability assayHepatocellular carcinoma cells treated in the presence

or absence of ODN were plated at a density of 1 � 104cells/well in 96-well plates. After 4 hours, NK-92,NKL, orprimaryNKcellswere added into the plates at different E:T values. At the 12-hour time-point, 10 mL (5 mg/mL)MTT (Sigma) was added and incubated for another 4hours. After centrifugation, 100 mL of the supernatantswere removed from each well, and 100 mL of 10% SDSsolution was added to dissolve the formazan crystals andincubated at 37�C, in 5% CO2 overnight. Then, the absor-bance at 570 to 630 nmwas determined using aMicroplateAutoreader (Bio-Rad).

Cell cytotoxicity assayCell cytotoxicity againstHepG2 cellswas evaluated in a

4-hourCFSE/7-AADflowcytometry assay.After labelingwith 5-6-carboxyfluo-rescein diacetate succinimidyl ester(CSFE, Beyotime) for 15 minutes at 37�C, CFSE-labeledhepatocellular carcinoma cells were washed with medi-um and seeded in completemedium for adherent culture.Then, NK-92 cells were added with E:T at 5:1. Hepato-cellular carcinoma cells were incubated alone to measurebasal cell death. After 4 hours, cells were collected andwashed twice with 1� PBS and incubated with 7-aminoactinomycin D (7-AAD, KeyGEN BioTECH) for 15 min-utes at room temperature in darkness. Acquisition wasperformed with a FACSCalibur system (BD Biosciences).The following formulawas used to calculate specific lysis.Ratio ¼ % CFSEþ 7-AADþ/% CFSEþ,% specific lysis ¼(the ratio of sample � ratio of basal) � 100

Statistical analysisAll data are presented as the means � SD of three or

more independent experiments. Statistical analysis wasperformed using a paired Student t test. P < 0.05 wasconsidered statistically significant.

ResultsBlocking STAT3 in hepatocellular carcinomaaugmented the susceptibility of hepatocellularcarcinoma to NK-cell cytolysis

We have previously shown that blocking STAT3 sup-pressed the growth and promoted the apoptosis of hepa-tocellular carcinomacells (13). The resistance of tumor cellsto NK cell cytolysis contributed to antitumor immunesuppression. Therefore, we investigated the effects ofblocking activated STAT3 on the sensitivity of hepatocel-lular carcinoma cells to NK cell cytolysis. In Fig. 1A and B,human NK cell lines NK-92 and NKL as well as humanPBMCswereusedaseffector cells, respectively.Comparedwith theLipo-Ctrl (Lipofectamine reagent control) groups,the viability of decoy ODN-treated hepatocellular carci-noma cells exposed toNK cells for 12 hourswas decreasedsignificantly, with minimum viability of 41% to 18%. Inaddition, NK-92-cell–mediated specific cell lysis againsthepatocellular carcinoma cells was evaluated in a 4-hourCFSE/7-AAD flow cytometry assay. As shown in Fig. 1C,NK-92 cell cytotoxicity against decoy ODN-treatedhepatocellular carcinoma was significantly higher at5:1 E:T ratios in comparison with Lipo-Ctrl groups. Incontrast, scramble ODN treatment did not influence theviability or sensitivity of hepatocellular carcinoma toNK cell lysis. These data indicated that blocking STAT3in hepatocellular carcinoma enhanced the sensitivity ofhepatocellular carcinoma cells to NK cell–mediatedcytolysis.

The NKG2D ligands expressed on hepatocellularcarcinoma cellswere upregulated by blocking STAT3

As a result of "genomic stress", NKG2D ligands, whichare rarely detectable on the surface of healthy cells and

Blocking STAT3 in Hepatocellular Carcinoma Cells Restored NK Activation

www.aacrjournals.org Mol Cancer Ther; 12(12) December 2013 2887




tissues, are upregulated in tumor cell lines and tumorissues, rendering the tumor cell susceptible to NK cellcytotoxicity (23). However, cancer cells have also devel-oped strategies to evade NKG2D-mediated responses.Cumulative evidence indicates that the expression levelsof NKG2D ligands on tumor cells are important in NK-cell–mediated antitumor effects. Therefore, we examinedchanges in the expression of NKG2D ligands in STAT3-blocked hepatocellular carcinoma cells. As shown in Fig.2A, blockingSTAT3caused significantupregulation in theexpression levels of ULBPs, especially ULBP3. Althoughthe experimental findings showed that STAT3 directlyinteracts with the MICA promoter to repress MICA tran-scription in colorectal cancer (21), there were no obviouschanges in the levels of MICA/B in HepG2 cells treatedwith STAT3-decoy ODN compared with that in Lipo-Ctrlcells. To further determine the function of NKG2Dligands, an anti-hULBP3 mAb (40 mg/mL) was used toblock ULBP3 expressed on HepG2 cells, and the viabilityof these tumor cells in the presence of NK cells wasmeasured (Fig. 2B). ULBP3 blockade significantlyenhanced the viability of STAT3-blocked hepatocellularcarcinoma cells, suggesting that upregulation of NKG2Dligands, especiallyULBP3, contributed to the sensitivity ofSTAT3-blocked hepatocellular carcinoma cells to NK-cellcytotoxicity.

Blocking STAT3 reversed hepatocellular carcinoma-induced immune suppression on NK cells.

Impairment of NK cell function contributes to antitu-mor immune inefficiency in patients with hepatocellularcarcinoma (5), which is partly mediated by immunosup-pressive factors secreted by tumor cells. Then, to clarifywhether hepatocellular carcinoma-mediated NK cell dys-function could be recovered by blocking STAT3, humanNK cells were incubated with the supernatants fromhuman hepatocellular carcinoma cells treated with orwithout STAT3-decoy ODN for 12 hours, and the influ-ence of these NK cells on the viability of hepatocellularcarcinoma cells was then examined. As shown in Fig. 3A,after incubation with supernatants from the Lipo-Ctrl orscramble ODN-treated hepatocellular carcinoma cells,NK cell–mediated anti-hepatocellular carcinoma effectwas suppressed compared with that of untreated NKcells (Medium); however, it was enhanced when NK cellswere incubated with the supernatant from hepatocellularcarcinoma cells treated with STAT3 decoy ODN, withenhanced levels exceeding those mediated by untreatedNK cells (Medium). Similar results were observed whenhuman PBMCswere used as effector cells. The viability ofHepG2 cells exposed to healthy PBMCs was 48.95% �0.32%, which was decreased to 30.37% � 0.53% in thepresence of PBMCs treated with the supernatant from

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Figure 1. Blocking STAT3 in hepatocellular carcinoma augmented the susceptibility of hepatocellular carcinoma cells to NK cell cytolysis. As describedin the Materials and Methods section, hepatocellular carcinoma cells were transfected with STAT3 decoy ODN, scramble ODN, or Lipofectaminereagent control (Lipo-Ctrl) and used as target cells. The viabilities of these cells in the presenceofNK-92/NKL cells (A) andhumanPBMCs (B)were detected byMTT assay with different E:T ratios. C, the sensitivities of these cells to NK-92 cell cytolysis were evaluated in a 4-hour CFSE/7-AAD flow cytometryassay with an E:T ratio of 5:1. Data are representative of three independent experiments, and statistical significance was determined as �, P < 0.05 and��, P < 0.01 compared with the Lipo-Ctrl.

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STAT3-blocked HepG2 cells (Fig. 3B). These observationswere confirmed using NSC74859, a chemical probe inhib-itor of STAT3 activity to block STAT3 in hepatocellularcarcinoma cells (Supplementary Fig. S1). These findingsindicated that blocking STAT3 in hepatocellular carcino-ma reversed hepatocellular carcinoma-mediated immune

suppression and promoted NK-cell–mediated antitumoreffects.

NK cells were activated by the products secreted bySTAT3-blocked hepatocellular carcinoma cells

NK cell cytolysis is determined by the balance betweeninhibitory and activatory signals. When activated, NKcells attack a target via manymechanisms, such as releaseof cytolytic granules (e.g., perforin and granzymes), anti-body-dependent cell-mediated cytotoxicity (ADCC), pro-duction and secretion of cytokines (e.g., IFN-g , TNF-a;ref. 24), as well as evasion of "self" by downregulation ofMHC-I molecules (25). In order to investigate the char-acteristics of NK cells treated as described, total mRNA ofNK-92 cells was extracted and the molecules associatedwithNKactivationwere analyzedbyqRT-PCR.As shownin Fig. 4A and Supplementary Fig. S2A, compared withthe Lipo-Ctrl and Scramble groups, we found that mRNAlevels ofmolecules related to cytolysis, includingNKG2D,IFN-g , FasL, perforin, granzyme B, and TNF-a, wereincreased obviously in NK-92 cells incubated with thesupernatant from STAT3-blocked hepatocellular carcino-ma cells, as well the protein levels detected by ELISAand flow cytometric analysis (Fig. 4B and 4C and Sup-plementary Fig. S2B). However, the protein level ofNKG2A was decreased in NK-92 cells by treatmentwith the supernatant from STAT3 decoy ODN-treatedhepatocellular carcinoma cells (Fig. 4C and Supplemen-tary Fig. S2B). Furthermore, as shown in Fig. 4D, similarresults were observed when NK-92 cells were replacedwith human primary CD3�CD56þ PBMCs, and thelevels of activation-related molecules—CD69, NKG2D,and perforin—on these cells increased obviously com-pared with the Lipo-Ctrl and Scramble groups. Thesefindings demonstrated that NK cells were activated asSTAT3 in hepatocellular carcinoma cells was blocked,and implicated that the products secreted by STAT3-blocked hepatocellular carcinoma cells in the underly-ing mechanisms.

The cytokine profile of hepatocellular carcinomacells was changed by blocking STAT3

In the tumor microenvironment, tumor cells suppressimmune surveillance by secreting proinflammatory cyto-kines and immunosuppressive cytokines (26, 27). There-fore, we aimed to determine the effects of blocking STAT3on the cytokine profile of hepatocellular carcinoma cells.As shown in Fig. 5A, in STAT3-blocked hepatocellularcarcinoma cells, the mRNAs levels of inflammatory cyto-kines, including IL-6, -8, -18, -17, and -23, as well as theimmunosuppressive factor TGF-b were downregulated,whereas the mRNA levels of IFN-a, -b, and -g wereupregulated. In the tumor microenvironment, IL-8, IL-10, and TGF-b are known to function as immunosuppres-sive factors and exert positive effects on tumor cells (28,29). ELISA analysis of the protein levels showed lowerlevels of IL-8, IL-10, andTGF-b inHepG2 cells treatedwiththe STAT3 decoy ODN (Fig. 5B). Similar results were also


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Figure 2. The NKG2D ligands expressed on hepatocellular carcinomacells were upregulated by blocking STAT3. A, twenty-four hours aftertransfection with STAT3-decoy ODN, scramble ODN, or Lipofectaminereagent control (Lipo-Ctrl), the expression of NKG2D ligands and Fas onHepG2 cells was determined by flow cytometric analysis. Data arerepresentative of three independent experiments, and statisticalsignificance was determined as P < 0.05 (�) and P < 0.01 (��) comparedwith the Lipo-Ctrl. B, STAT3 decoy ODN-treated HepG2 cells wereincubated with anti-hULBP3 mAb (30 mg/mL; Decoyþa-ULBP3) orimmunoglobulin G (DecoyþIgG) for 1 hour before assay of the viability inthepresenceofNK-92cells. Data are representativeof three independentexperiments; statistical significance was determined as P < 0.05 (�) andP < 0.01 (��) compared with the indicated group. NS, no significance.






found in the other human hepatocellular cell lines H7402and PLC/PRF/5.

TGF-b1 reduction and type I IFN production bySTAT3-blocked hepatocellular carcinoma cells werecritical for NK-cell activation

As a multifunctional cytokine, TGF-b plays importantroles in tumor immune evasion (30). It is overproduced inthe serum of patients with cancer and is linked withreduced NK-cell activity (3). Furthermore, IL-10, whichis also associated with tumor malignancy via immuneescape, is involved in the phosphorylation of JAK2/STAT3 (8). In order to determine whether the productionof TGF-b1 and IL-10 was responsible for the suppression

in NK-cell function, first, the regulatory effects of STAT3on the transcriptional activities of the TGF-b1 and IL-10promoters were identified by a dual-luciferase assay (Fig.6Aand6B).Next,NK-92 cellswere incubatedwithHepG2cell supernatant with or without a TGF-b1 neutralizingantibody, and the effects on the viability of HepG2 cellswere evaluated. The results showed that the viability ofHepG2 cells in the presence of NK-92 cells incubatedwithHepG2 cell supernatant was 52.42% � 1.09%, which wasdecreased to 40.63% � 0.55% in the presence of theneutralizing TGF-b1 (Fig. 6C). The viability of HepG2cells in the presence of NK-92 cells incubated with thesupernatant from STAT3 decoy ODN-treated hepatocel-lular carcinoma cells was 22.05% � 2.62%, which was

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Figure 3. Blocking STAT3 inhepatocellular carcinoma cellstriggered the antitumor effects ofNK cells. A and B, afterhepatocellular carcinoma cellswere transfected with STAT3-decoy ODN, scramble ODN, orLipofectamine reagent (Lipo-Ctrl)as described in the Materials andMethods section, the supernatantswere collected and used to cultureNK-92/NKL cells (A) or humanPBMCs (B) for 12 hours. Theinhibitory effect of theseNK cells orPBMCs on the viability of untreatedhepatocellular carcinoma wasanalyzed by MTT assay. Data arerepresentative of threeindependent experiments;statistical significance wasdetermined as P < 0.05 (�) andP < 0.01 (��) compared with theLipo-Ctrl.

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reversed by the addition of TGF-b1. In addition, flowcytometry assay with CFSE/7-AAD was employed toevaluate NK-92-cell–mediated specific cell lysis againsthepatocellular carcinoma cells, and consistent findingswere observed (Fig. 6D). In contrast, scramble ODN treat-mentdidnot influence theNK-92 cell cytolysis. Thesedataindicated that TGF-b1 acted as an important factor in thehepatocellular carcinoma-mediated immunosuppressiveeffects on NK cells.Nevertheless, as shown in Fig. 6C and 6D, the anti-

hepatocellular carcinoma effects of NK cells treated withhepatocellular carcinoma supernatant containing anti-TGFb1 mAb was still lower than that of NK-92 cellsincubated with supernatant from the STAT3 decoyODN-treated hepatocellular carcinoma cells. We specu-lated that some activating cytokines are induced in tumorcells by STAT3 blockade, which stimulated the cytolyticcapacity of NK cells. The levels of IFN-a, -b, and -g wereupregulated (Fig. 5A), similarly as the p-STAT1 levels andtranscriptional activity of STAT1 in HepG2 cells beingincreasedby STAT3decoyODN(Fig. 7A). Investigation of

the role of interferons by antibody neutralization showedthat anti-IFNAR mAb suppressed the anti-hepatocellularcarcinoma activities of NK cells induced by the superna-tant from STAT3-blocked hepatocellular carcinoma cells(Fig. 7B and 7C). Concomitant downregulation of mole-cules associated with NK activation was observed (Fig.7D). Furthermore, the cytolytic activity of NK cells wasrestored by the addition of type I IFN added to thesupernatant of hepatocellular carcinoma cells (from18.24% � 1.1% to 33.57% � 1.65%; Fig. 7C). These resultsdemonstrated that both TGF-b1 reduction and type I IFNproduction were essential for blocking STAT3-inducedNK-cell activation.

DiscussionAs an oncogene, aberrant STAT3 activation has been

detected in many tumors, controlling differentiation, pro-liferation, survival, angiogenesis, and immune functionduring tumorigenesis (15). Furthermore, increasing evi-dence demonstrates that tumor-induced abnormalities in

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Figure 4. NK cells were activated by the products secreted by STAT3-blocked hepatocellular carcinoma cells. The supernatants, collected from HepG2 cellstreated with STAT3-decoy ODN, scramble ODN, or Lipofectamine reagent (Lipo-Ctrl) as described in the Materials and Methods section, were used toincubate NK-92 cells for 12 hours. The molecules associated with NK-cell cytolysis were analyzed by qRT-PCR (A), ELISA (B), and flow cytometry (C).D, human PBMCs were incubated with the supernatants from HepG2 cells treated as described earlier. Twelve hours later, the levels of molecules related toNK-cell activation and cytolysis were analyzed by flow cytometry. The histogram represents statistical analysis of the percentage of positive cells. Data arerepresentative of three independent experiments; statistical significance was determined as P < 0.05 (�) and P < 0.01 (��) compared with the Lipo-Ctrl.






the immune system promote tumor progression andimmune evasion. Therefore, a better identification andunderstanding of the factor(s) involved in this abnormal-ity is the crux of this problem.

In a previous study, we observed that blocking STAT3activation efficiently inhibited the growth of hepatocellu-lar carcinoma cells (13). Nevertheless, aberrant activationof STAT3 is closely related with immunosuppression andtumor evasion (14–16). Therefore, in this study, we aimedto determine whether targeted blockade of STAT3 inhuman hepatocellular carcinoma cells would break hepa-tocellular carcinoma-mediated antitumor immune sup-pression and improve the function of NK cells.

Decoy ODNs, specifically designed to compete withendogenous cis-elements of the target gene, have beenproposed as a useful approach to block the function oftranscription factors. Decoy methods exhibit severalattractive advantages over other therapeutic strategies.First, decoyODNs, as small DNAmolecules, can be easilydelivered to target tissues and transfected into cells, anddirectly abrogate the activated transcript factors. Second,a growing number of transcription factors and their pro-moter sequences have been characterized, which makespotential drug targets plentiful and readily identifiable.Third, the synthesis, storage, and transportation of decoyODNs are much simpler than other approaches. More-over, decoy ODN strategies have been widely used inthe laboratory and clinical studies of many diseases(11, 13, 19, 31). In this study, by fluorescence microscopy(Supplementary Materials and Methods), the subcellulardistribution of STAT3 decoy ODN in HepG2 cells was

shown to be mainly located in nucleus and lasted atleast 48 hours after transfection (Supplementary Fig.S3). In addition, this was observed when using scrambleODN.

Our results showed that blocking STAT3 in hepatocel-lular carcinoma augmented the susceptibility of hepato-cellular carcinoma to NK-cell–mediated cytolysis (Fig. 1).The activatory and inhibitory receptors present on NKcells are triggered during target cell recognition andinduce a positive or a negative cell signaling pathway,respectively; in addition, the balance between activatoryand inhibitory signals determines the activation of NKcells. When activated, NK cells eliminate their targetthrough the release of cytotoxic enzymes (perforin, gran-zymes, granulysin) and/or soluble factors (chemokinesand inflammatory cytokines), which, in turn, recruit and/or activate other effectors, such as promoting macro-phages phagocytosis and CD8þ T-cell cytotoxicity (24).In contrast, inhibitory receptors on NK cells limit exces-sive activation and regulate immune responses. Thesemolecules are the targets of evasion strategies exerted bytumors. For instance, in a breast tumor, impaired NK-cellfunction correlated with decreased activatory NK cellreceptors (such as NKp30, NKG2D, DNAM-1, and CD16)and increased inhibitory receptors (such as NKG2A) (3).In the present study, we found that NK cells were acti-vated by incubation with the supernatant from STAT3-blocked hepatocellular carcinoma cells and accompaniedby enhancement of perforin, granzymes, IFN-g , NKG2D,and CD69, whereas the inhibitory receptor (NKG2A) wasdecreased (Fig. 3 and 4).

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Figure 5. The cytokine profile inhepatocellular carcinoma cells waschanged by STAT3 decoy ODN.After transfection with STAT3decoy ODN, scramble ODN, orLipofectamine reagent (Lipo-Ctrl)for 24 hours, HepG2, H7402, orPLC/PRF/5 cells were harvestedand the levels of tumor-relatedinflammatory cytokines wereexamined by qRT-PCR (A) andELISA (B) analysis. Data arerepresentative of threeindependent experiments;statistical significance wasdetermined as P < 0.05 (�) andP < 0.01 (��) compared withthe Lipo-Ctrl.

Sun et al.





The recognition between activatory receptors of NKcells and ligands expressed on cancer cells is an importantmechanism by which NK cells eliminate targets. Amongthe major activatory receptors expressed on NK cells,NKG2D is implicated in the surveillance of viral infectionand cancer. Despite DNA damage or heat-shock proteins,which would actively upregulate the levels of NKG2Dligands on cancer cells, many tumors have been shown toinhibit NKG2D ligand expression by the production ofimmunomodulatory cytokines (21, 32–34). Hilpert andcolleagues demonstrated that the antitumor activitymediated byNK cells was greatly dependent on the levelsofNKG2D ligands expressed on the surface of tumor cells,including MICA/B and ULBP1-3 (35). In this study, we

found that the expression levels of ULBPs, especiallyULBP3, were obviously upregulated in hepatocellularcarcinoma cells by STAT3 blockade (Fig. 2A), which wasfavorable for NK-cell recognition and enhanced the sus-ceptibility of hepatocellular carcinoma to NK-cell cytoly-sis. Furthermore, our results showed that the cytolyticactivity of NK cells against decoy ODN-treated hepato-cellular carcinoma cells was suppressed by blocking theexpression of ULBP3 (Fig. 2B). Although Bedel and col-leagues demonstrated that STAT3 modulated MICAexpression in cancer cells (21), the expression of MICA/B in hepatocellular carcinoma was not changed signifi-cantly by blocking STAT3. It can be speculated that this isbecauseNKG2D ligands are stress-response genes andare

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Figure 6. TGF-b1 reduction by STAT3 decoy ODN-treated hepatocellular carcinoma cells were critical for NK-cell activation. A and B, HepG2 cells werecotransfected with pGL3-TGFb1-Promoter-Luciferase, pGL3-IL10-Promoter-Luciferase, or pGL3-TK-Luciferase and STAT3-decoy or scramble ODN in thepresence of Lipofectamine 2000, whereas the Renilla expression vector pRLTK was cotransfected to normalize the transfection efficiency. After 24 hours,luciferase activity of lysed cells was measured using a dual-Glo Luciferase assay system. The ratio of firefly and Renilla luciferase activity associatedwith pGL3-TK-Luciferase transfection was set as 1. C and D, NK-92 cells were cultured with the supernatants from HepG2 cells supplemented with orwithout anti-TGF-b1 mAb (2 mg/mL) and the supernatants from STAT3 decoy ODN-treated HepG2 cells in the presence or absence of TGF-b1 (2.5 ng/mL)for 12 hours. Subsequently, the inhibitory effect of these NK-92 cells on HepG2 cell viability was detected by MTT assay (C), and the specific lysis ofthese NK-92 cells against HepG2 cells was detected by a 4-hour CFSE/7-AAD flow cytometry assay with E: T at 5:1 (D). Medium, NK cells cultured in ana-MEM without any treatment; IgG þBSA, NK cells treated with IgG and BSA to exclude the effects of IgG þBSA; Lipo-Ctrl, NK cells cultured with thesupernatants fromLipofectamine reagent-treatedHepG2 cells; Scr, NKcells culturedwith the supernatants from scrambleODN-treatedHepG2 cells; Scrþa-TGFb1, NK cells culturedwith the supernatants from scramble ODN-treated HepG2 cells supplementedwith anti-TGF-b1mAb; Decoy, NK cells culturedwiththe supernatants from STAT3 decoy ODN-treated HepG2 cells; DecoyþTGFb1, NK cells cultured with the supernatants from STAT3 decoy ODN-treatedHepG2 cells supplemented with TGF-b1. Statistical significance was determined as P < 0.05 (�) and P < 0.01 (��); NS, no significance.






upregulated by stimuli such as DNA damage and heat-shock. Thus, the high basic level of MICA/B in hepato-cellular carcinoma may be the result of the oncogenicprocess itself. These data confirmed that blocking STAT3in hepatocellular carcinoma cells could reverse the hepa-tocellular carcinoma-mediated inhibitory effects on NKcells through augmenting the expression of activatoryreceptors on NK cells and corresponding ligands onhepatocellular carcinoma cells, resulting in the enhance-ment of NK cell–antitumor functions.

Many factors regulated by STAT3 in tumor cells par-ticipate in promoting malignant cell proliferation, migra-tion, and invasion. In STAT3-targeted genes, IL-6, IL-8, IL-10, VEGF, and TGF-b disturb antitumor immune surveil-lance and result in tumor immune escape (8, 10, 28).Therefore, we tried to identify which cytokines are majorfactors involved in the regulation of NK-cell cytolyticactivity during STAT3 decoy ODN treatment. First, wefound that the cytokine profile of hepatocellular carcino-ma cells was changed by blocking STAT3, with the reduc-tion of inflammatory cytokines (IL-6, -8, -10, -17, and -23)and the immunosuppressive factor TGF-b, whereas stim-ulatory IFN was increased (Fig. 5). Cumulative studiesshowed thatmodulatory cytokines from the tumormicro-environment are responsible for the resistance of tumorcells to immune effectors (15). The immunosuppressivecytokine TGF-b and IL-10 play key roles in tumor immune

evasion. The levels of TGF-b are often elevated in theserum of patients with cancer, which is responsible forsuppressing the activation of NK cells and cytotoxic Tcells, as well as the differentiation of regulatory T cells(1, 31). By blocking STAT3 in hepatocellular carcinomacells, we found that levels of both TGF-b1 and IL-10 weredecreased at the protein andmRNA levels (Fig. 5), accom-panied by downregulation of TGF-b1 and IL-10 transcrip-tional activities (Fig. 6A and 6B). In addition, it has beenreported that activated STAT3 directly binds to the IL-6promoter to positively regulate IL-6 expression (36),which is in accord with our observation that IL-6 mRNAwas downregulated by blocking STAT3. In contrast, as astimulator of innate and adaptive immune responses,the mRNA of IFNs in hepatocellular carcinoma wasincreased by STAT3 blockade (Fig. 5A). Type I IFNactivates NK, DCs, CD4 and CD8 T cells, plays a centralrole in the process of antitumor immune responses andinduces STAT1 activation (37, 38). In addition, IFN-a/bhas been considered as a strategy for tumor immuno-chemotherapy through upregulation of NKG2D ligandsand MHC class I, leading to the growth inhibition oftumor cells (39). Our data presented in Fig. 7A, showsthat p-STAT1 was upregulated by STAT3 decoy treat-ment in HepG2 cells, and the cytolytic activity of NKcells was inhibited by anti-IFNR neutralizing antibody(Fig. 7B and 7C).

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Figure 7. Type I IFN production by STAT3 decoyODN-treated hepatocellular carcinoma cells was critical for NK-cell activation. A,Western blotting analysis ofphospho-STAT1 in lysates prepared fromHepG2 transfected with STAT3 decoy ODN, scramble ODN, or Lipofectamine reagent (Lipo-Ctrl) for 24 hours (left).Luciferase reporter assay was used to determine the transcriptional activity of STAT1 with STAT3-decoy or scramble ODN transfection (right).B to D, NK-92 cells were cultured with the supernatants from scramble ODN-treated-HepG2 cells supplemented with or without IFN-a (400 U/mL) þ IFN-b(400 U/mL; ScrþIFN and Scr, respectively), and the supernatants from STAT3 decoy ODN-treated HepG2 cells in the presence or absence of anti-IFNAR(10 mg/mL; Decoyþa-IFNR and Decoy, respectively) for 12 hours. Subsequently, the suppressive effect of these NK-92 cells on HepG2 cell viability (B)and the specific lysis of NK-92 cells against HepG2 cellswere detected (C), and expressions of themolecules associatedwithNK-cell cytolysis were analyzedby flow cytometry (D). Data are representative of three independent experiments; statistical significance was determined as P < 0.05 (�) and P < 0.01 (��)compared with the indicated groups; NS, no significance.

Sun et al.





In conclusion, this study demonstrated that hepato-cellular carcinoma cells create a multifaceted immuno-suppressive microenvironment that suppresses NK-cell functions and, furthermore, that blocking STAT3in hepatocellular carcinoma cells restored NK-cell–

mediated antitumor efficiency. As shown in Fig. 8,TGF-b reduction and type I IFN production were bothcritical to NK-cell activation, and the upregulation ofNKG2D ligands in hepatocellular carcinoma cellsfacilitated NK-cell recognition and activation. Thesefindings indicate that targeted blockade of STAT3 inhepatocellular carcinoma cells not only promotes tumorcell apoptosis directly but also released hepatocellularcarcinoma-induced antitumor immune suppression ofNK cells indirectly, suggesting a new anti-hepatocellu-lar carcinoma immunotherapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: X. Sun, Q. Sui, C. Zhang, Z. Tian, J. ZhangDevelopment of methodology: X. Sun, Q. Sui, J. ZhangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Sun, Q. Sui, J. ZhangAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): X. Sun, Q. Sui, C. Zhang, J. ZhangWriting, review, and/or revisionof themanuscript:X. Sun,Q. Sui, Z. Tian,J. ZhangAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): X. Sun, Q. Sui, C. Zhang, J. ZhangStudy supervision: Z. Tian

AcknowledgmentsThe authors thank the editor and three anonymous referees for their

constructive advice and comments to improve this work.

Grant SupportThisworkwasfinancially supported by theNatural Science Foundation

of China (grants no. 81172789, 30972692, and 30628014; to J. Zhang).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 15, 2012; revised July 29, 2013; accepted August 14,2013; published OnlineFirst October 9, 2013.

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NK

HCC

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Apoptosis

TGF-b, IL-10

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Anti-HCC effects

PerforinGZMIFN-g …

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Immunosuppresive molecules

Cytolysis

+

STAT3 decoy ODN

Figure 8. Targeted blockade of STAT3 in hepatocellular carcinoma (HCC)cells augmented NK cell function. Blocking STAT3 in hepatocellularcarcinoma induced tumor apoptosis directly while NK cell antitumorfunction was enhanced indirectly. As shown, expression of NKG2Dligands by hepatocellular carcinoma cells was upregulated with STAT3blockade, which promoted the recognition by NK cells and enhanced thesusceptibility of hepatocellular carcinoma cells to NK cell cytolysis.Furthermore, the cytokine profile of hepatocellular carcinoma cells wasaltered, resulting in the reduction of immunosuppressive molecules,especially TGF-b and IL-10, and the induction of type I IFN. All thesechanges resulted in the release of the hepatocellular carcinoma-inducedimmunosuppressive status, and the cytotoxicity of NK cells againsthepatocellular carcinoma cells was enhanced.






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Sun et al.





2013;12:2885-2896. Published OnlineFirst October 9, 2013.Mol Cancer Ther Xiaoxia Sun, Qiangjun Sui, Cai Zhang, et al. Induced Immune Suppression−

Augments NK Cell Functions via Reverse Hepatocellular Carcinoma Targeting Blockage of STAT3 in Hepatocellular Carcinoma Cells

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targeting blockage of stat3 in hepatocellular carcinoma ...ifn derived from stat3-blocked...

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